Quantification of retinal transneuronal degeneration in human glaucoma: a novel multiphoton-DAPI approach.

PURPOSE: Glaucoma is presumed to result in the selective loss of retinal ganglion cells. In many neural systems, this loss would initiate a cascade of transneuronal degeneration. The quantification of changes in neuronal populations in the middle layers of the retina can be difficult with conventional histologic techniques. A method was developed based on multiphoton imaging of 4',6'-diamino-2-phenylindole (DAPI)-stained tissue to quantify neuron loss in postmortem human glaucomatous retinas. METHODS: Retinas from normal and glaucomatous eyes fixed in 4% paraformaldehyde were incubated at 4 degrees C overnight in DAPI solution. DAPI-labeled neurons at different levels of the retina were imaged by multiphoton confocal microscopy. Algorithms were developed for the automated identification of neurons in the retinal ganglion cell layer (RGCL), inner nucleus layer (INL), and outer nuclear layer (ONL). RESULTS: In glaucomatous retinas, the mean density of RGCs within 4 mm eccentricity was reduced by approximately 45%, with the greatest RGC loss occurring in a region that corresponds to the central 6 degrees to 14 degrees of vision. Significant neuron loss in the INL and ONL was also seen at 2 to 4 mm and 2 to 3 mm eccentricities, respectively. The ratios of neuron densities in the INL and ONL relative to the RGCL (INL/RGC and ONL/RGC, respectively) were found to increase significantly at 3 to 4 mm eccentricity. CONCLUSIONS: The data confirm that the greatest neuronal loss occurs in the RGCL in human glaucoma. Neuronal loss was also observed in the outer retinal layers (INL and ONL) that correlated spatially with changes in the RGCL. Further work is necessary to confirm whether these changes arise from transneuronal degeneration.

T2 weighted MRI for assessing renal lesions in transgenic mouse models of tuberous sclerosis.

OBJECTIVE: Transgenic mouse models of tuberous sclerosis (TSC) develop renal cysts, cystadenomas, solid adenomas and carcinomas. Identification and characterisation of these lesions in vivo may help in TSC pre-clinical trials. This study was to evaluate T2 weighted MRI for assessment of renal lesions in two Tsc mouse models. MATERIALS AND METHODS: Tsc1(+/-), Tsc2(+/-) and wild type mice were subjected to a first MRI scan at 12 months of age and a second scan 2 months later. One Tsc2(+/-) mouse was treated with rapamycin for two months after the initial scan. Immediately following the second scan, mice were sacrificed and MRI images were compared to renal histological findings. RESULTS: MRI identified all types of Tsc-associated renal lesions in both Tsc1(+/-) and Tsc2(+/-) mice. The smallest detectable lesions were <0.1 mm(3). Eighty three percent of all renal lesions detected in the first scan were re-identified in the second scan. By MRI, these lesions demonstrated significant growth in the 9 untreated Tsc1(+/-) and Tsc2(+/-) mice but shrinkage in the rapamycin treated Tsc2(+/-) mouse. Between the two scans, MRI also revealed significant increase in both the total number and volume of lesions in untreated mice and decrease in the rapamycin treated mouse, respectively. In comparison to histological analysis MRI detected most cysts and cystadenomas (66%) but only a minority of solid tumours (29%). CONCLUSION: These results suggest that T2 weighted MRI may be a useful tool for assessing some renal lesions in pre-clinical studies using Tsc mouse models. However, improved sensitivity for T2 weighted MRI is required, particularly for solid renal lesions.

A validated method for quantifying macrovesicular hepatic steatosis in chronic hepatitis C.

Hepatic steatosis is increasingly seen as an important prognostic factor in chronic hepatitis C infection (HCV). The commonly used semiquantitative method of measuring steatosis is based on a study that excluded patients with HCV. Several potentially useful methods of quantifying steatosis using computer-assisted morphometric analysis have been proposed, but none has been validated against a proposed gold standard other than the method they were intended to replace. We present a novel method and propose a gold standard based on manual measurements. The manual method is time consuming but shows little interobserver error, and the mean value of 3 observations by separate investigators is proposed as the gold standard. The computer-assisted method is fast, with a single interactive step that shows minimal interobserver variation. It accurately identifies biopsies with <1% steatosis (7 of 7) and predicts the gold standard value for biopsies with > 1% steatosis with narrow CIs (geometric mean ratio 0.85 with 95% CIs 0.77-0.95). This novel method of computer-assisted morphometric analysis is fast, reliable, and suitable for future research in HCV steatosis. It may be used to reanalyze previous studies. The semiquantitative method of assessing steatosis remains appropriate for clinical purposes.

Effects of a single infusion of pamidronate prior to liver transplantation: a bone histomorphometric study.

Osteoporosis is a common and serious complication of solid organ transplantation. Effective therapeutic regimens have not been established but evidence that increased bone turnover is responsible for bone loss early after transplantation provides a rationale for the use of anti-resorptive agents in the peri-operative period. We have examined the effects of a single pre-operative infusion of pamidronate, 60 mg, on bone remodelling and turnover in a prospective study of 12 patients, four male and eight female aged 19-61 years, with chronic liver disease, who formed a subgroup of a larger randomised controlled single-blind study. Iliac-crest biopsies were obtained before and 3 months after liver transplantation and histomorphometry performed using image analysis. In untreated patients ( n=5) a significant increase in bone formation rate at tissue level was demonstrated at 3 months in comparison to pre-operative values (0.035+/-0.013 vs. 0.161+/-0.12 microm(2)/microm/day; mean +/- SD, P=0.003). In patients treated with pamidronate ( n=7) no significant increase in bone formation rate was demonstrated at 3 months, although there was a trend towards an increase in indices of bone turnover. In this group there was also a significant reduction in erosion cavity length (210.4+/-63.8 vs. 179.8+/-67.5 microm; P=0.03) and non-significant reductions in other indices of erosion cavity size. These results indicate that pre-operative administration of pamidronate in patients with chronic liver disease prevents, at least in part, the increase in bone turnover which occurs in untreated patients after transplantation.

Histomorphometric analysis of bone biopsies from the iliac crest of adults with cystic fibrosis.

This study reports the results of quantitative analysis of iliac bone histology in adults with cystic fibrosis (CF) and low bone mineral density (BMD). Twenty patients with CF had bone biopsies taken after double tetracycline labeling. Histomorphometric measurements were made by image analysis, and data were compared with those of healthy control subjects. Cancellous bone area was lower in the patients with CF (p = 0.003), and there was a trend towards a decrease in cancellous bone connectivity. Bone formation rate at tissue level was significantly lower in patients with CF (p = 0.0002). Wall width, representing the amount of bone formed within individual remodeling units, was decreased (p < 0.0001), as was mineralizing perimeter and mineral apposition rate. Analysis of resorption cavities revealed lower cavity area, reconstructed surface lengths, and cavity depths (p < 0.003) in patients with CF, whereas eroded surface area was higher (p = 0.0004). Our results demonstrate low cancellous bone volume in adult patients with CF with low BMD, the main cause of which appears to be low bone formation at tissue and cellular level. Osteomalacia was diagnosed in one patient. This condition should be excluded as a cause of low bone mineral density in patients with CF and vitamin D insufficiency corrected.